This invention relates generally to the silage process, to microorganisms, and use of the same in treating animal feed and silage to enhance aerobic stability, thus preventing spoilage.
The ensiling process is a method of moist forage preservation and is used all over the world. Silage accounts for more than 200 million tons of dry matter stored annually in Western Europe and the United States alone. The concept involves natural fermentation, where lactic acid bacteria ferment water soluble carbohydrates to form organic acids under anaerobic conditions. This causes a decrease in pH which then inhibits detrimental microbes so that the moist forage is preserved. The process can be characterized by four different phases.
Upon sealing in the storage unit, the first phase is aerobic, when oxygen is still present between plant particles and the pH is 6.0 to 6.5. These conditions allow for continued plant respiration, protease activity and activity of aerobic and facultative aerobic microorganisms.
The second phase is fermentation which lasts several days to several weeks after the silage becomes anaerobic. Lactic acid bacteria develop and become the primary microbial population thereby producing lactic and other organic acids, decreasing the pH to 3.8 to 5.0.
The third phase is stable with few changes occurring in the characteristics of the forage so long as air is prevented from entering the storage unit.
The final phase is feedout when the silage is ultimately unloaded and exposed to air. This results in reactivation of aerobic microorganisms, primarily yeast, molds, bacilli and acetic acid bacteria which can cause spoilage.
Aerobic instability is the primary problem in silage production. Even before storage units are open for feedout, silage can be exposed to oxygen because of management problems (i.e., poor packing or sealing). Under these types of aerobic conditions, rapid growth of yeast and mold cause silage to heat and spoil, decreasing its nutritional value.
Aerobic instability can be a problem even in inoculated silage that has undergone what would traditionally be considered a xe2x80x9cgoodxe2x80x9d fermentation phase, namely a rapid pH drop, and a low terminal pH. The yeast which contribute to instability in these conditions may be those which are tolerant of acid conditions and can metabolize the lactic acid produced by lactic acid bacteria during fermentation.
Management techniques that can be used to help prevent this condition involve using care to pack the silage well during the ensiling process and, also, using care in removing silage for feeding to minimize the aeration of the remaining silage.
Management (compaction, unloading rates) largely affects the movement of oxygen into silage. During feedout, air can penetrate 1 to 2 m behind the silage face so that exposure to oxygen is prolonged. Fermentation acids and pH inhibit the rate of microbial growth, but spoilage rates are affected also by microbial numbers and the rate of aerobic microbial growth on available substrates.
It is possible to use both chemical and biological additives in making silage to promote adequate fermentation patterns especially under sub-optimal conditions. Biological additives comprise bacterial inoculants and enzymes. Bacterial inoculants have advantages over chemical additives because they are safe, easy to use, non-corrosive to farm machinery, they do not pollute the environment and are regarded as natural products. Silage inoculants containing principally homofermentative lactic acid bacteria have become the dominant additives in many parts of the world. Their function is to promote rapid and efficient utilization of a crop""s water soluble carbohydrates resulting in intensive production of lactic acid and a rapid decrease in pH. Inoculants also reduce aerobic spoilage and improve animal performance.
Several problems, however, with lactic acid bacteria inoculants have been encountered. These problems primarily include failure to dominate fermentation and failure to inhibit adverse microbial activity. Other problems associated with lactic acid bacteria inoculants include infection by phage, failure to grow well on certain crops, bacteria not being viable at the time of application, and the epiphytic nature of the lactic acid bacteria population. Because these types of homofermentative lactic acid bacteria inoculants do not always prevent or reduce undesirable microbial activity, several new approaches have been tried.
A review of the silage process and the use of inoculants can be found in FMS Microbiology Rev. 19 (1996) 53-68, Weinberg, ZNG., and Muck, R E, xe2x80x9cNew trends and opportunities in the development and use of inoculants for silagexe2x80x9d, the disclosure of which is incorporated herein by reference.
The concept of heterofermentative lactic acid bacteria in an inoculant has gained recent favor. The idea is that increased levels of undissociated volatile fatty acids, such as acetate, may inhibit other microbes that initiate aerobic deterioration. Heterofermenters have the ability to convert lactic acid to acetic acid in the presence of oxygen, and the acetate produced may inhibit other deleterious organisms. With such a mechanism, one-third of the lactic acid dry matter consumed will be lost as carbon dioxide. However a small loss of 1% or perhaps up to 2% dry matter may easily offset much larger losses by aerobic microorganisms. Concerns with heterofermentative lactic acid bacteria include effects on animal performance as well as the identification of appropriate strains useful for the procedure. Different strains of even the same species do not have identical properties and vary in their fermentation characteristics.
PCT publication WO 97/29644 discloses a single strain of Lactobacillus buchneri (NCIMB 40788) which was found to inhibit the growth of spoilage organisms in the storage of silage. Other attempts to identify heterofermentative organisms for silage inoculants have included (Wyss et al., 1991, xe2x80x9cEinfluss von Luftstress und die Wirkung von spezifishen Zusatzen anf die arobe Stabilitat von Grasswelksilagenxe2x80x9d, Wirschaftseigene Futter, 37: 129-141), which used an inoculant comprising lactate and propionate producing organisms in wilted grass silage. Weinberg et al. (1995), xe2x80x9cThe effect of a propionic acid bacterial inoculant applied at ensiling, with or without lactic acid bacteria on the aerobic stability of Pearl-Millet and maize silagesxe2x80x9d, J. Appl. Bacteriol., 78:430-436 disclosed the use of Propionibacterium shermanii in millet, corn, sorghum, and wheat silages. Propionic acid was produced only in a wheat silage in which the pH decline was delayed and thus aerobic stability was improved. In all other silages the pH decline was rapid and the propionic acid bacteria could not proliferate.
Another attempt included select strains of Serratia rubidaea and Bacillus subtilis along with L. plantarum. When used in bale grass silages the number of molds decreased significantly. Some improvement was also observed in high moisture ear corn. (Moran et al., (1993), xe2x80x9cThe development of a novel bacterial inoculant to reduce mold spoilage and improve the silage fermentation in big bale silage. In: Silage Research 1993, Proceedings of the Tenth International Conference on Silage Research (O""Kiely, P., O""Connell, M. and Murphy, J., Eds.) pp. 85-86, Dublin City University, Ireland). A similar composition to that for bale grass silage was developed for wheat silage which added Pediococcus strains to the composition. Pediococcus is capable of fermenting pentose sugars which result from hemicellulose hydrolysis in wheat silages. In a single trial with wheat silage, no improvement in the aerobic stability was observed.
The ensiling process is a complex one and involves interactions of numerous different chemical and microbiological processes. Further, different silages and different methods of ensiling present a variety of different needs. As can be seen a need exists in the art for further improvement in compositions and methods to improve the aerobic stability of silage.
It is an object of the present invention to provide a method and composition which can be used as an inoculant to improve aerobic stability of silage.
It is yet another object to provide novel yeast strains which can be used in compositions for improving aerobic stability.
It is a further object of the present invention to increase dry matter recovery of silage by reducing aerobic spoilage.
It is yet another object of the invention to provide an inoculant which is safe and nonhazardous for an additive to silage.
It is a further object of the invention to provide a natural additive composition for silage.
It is yet another object of the invention to provide quality silage material as determined by temperature, pH, dry matter recovery, nitrogen profile, color and microorganism count.
Other objects of the invention will become apparent from the description of the invention which follows.
According to this invention, forage materials which are to be stored are treated with particular yeast strains (xe2x80x9ckillerxe2x80x9d yeast) which inhibit the growth of wild yeasts that cause aerobic deterioration. These yeast strains also do not utilize lactate thus further preserving forage quality and inhibiting or retarding aerobic deterioration.
The invention provides a method of treating forage materials to enhance their preservation which comprises administering to the forage materials an effective amount of a killer yeast strain, its functional equivalents, or the forage preserving, or cytotoxic compositions produced thereby. According to the invention several strains of microorganisms have been identified from 18S rRNA sequencing to be from the class Saccharomyces exiguus, namely SE24, SE136 and SE151 which have been deposited with the ATCC, accession numbers 74441, 74442, 74443 respectively. Any one of these strains or any combination of the same may be used according to the invention.
As explained in more detail below, the microorganisms of the invention have a unique effect, different from and/or extending beyond their non lactate assimilation. The organisms produce antimicrobial factors likely proteinacious which are characterized by their ability to inhibit the growth of other yeast strains associated with the spoilage of silage or a variety of other spoilage organisms.
These substances may be isolated and purified by methods known to those of ordinary skill in the art. As such, the substances themselves may be used directly to treat animal feed or silage. In other words, it may not be necessary to use a microorganism as such in the compositions and methods of this invention.